Surgery and non-surgical anti-cancer therapies such as radiotherapy, chemotherapy, photodynamic therapy, immunotherapy, electric/chemotherapy, hyperthermia therapy, hyperbaric oxygen therapy, ischemia/reperfusion therapy and gene therapy have been found to be effective in the treatment of cancer. However, all of these treatments have been limited by tumor recurrence. In recent years, fundamental advances have been made in the development of regimens for solving these problems. Cancers continue to be the most common cause of death in countries throughout the world. The need for new and effective methods for treating cancer and leukemia continues to fuel efforts to find new classes of anti-tumor compounds or methods, especially for the inoperable or metastatic solid tumors, such as the various forms of lung cancer and hepatic carcinoma.
The characteristics and functions of cells are determined and maintained by cellular organelles and cellular cytoskeleton. Cellular organelles include nucleus, mitochondria, peroxisomes, Golgi apparatus, endoplasmic reticulum, centrosome, and vacules. Cytoskeletal structures (cytoskeleton) refer to an extensive scaffolding of fibrillar elements, including the three filamentous systems: microfilaments (or actin filaments), microtubules, and intermediate filaments. It may also include linin filaments. The components of the cytoskeleton are involved in diverse cellular functions ranging from mitosis to cell motility to signal transduction. In essence, the intact structure of the cytoskeleton and organelles constitutes the foundation of cell life, especially in the development of a variety of resistances. Among these organelles and cytoskeletal structures, the centrosome, microtubules, mitochondrion, and nuclear envelope are most important.
The centrosome, a central body (or the major microtubule organizing center (MTOC) of the cell, is composed of two centrioles surrounded by the so-called pericentriolar material (PCM), which consists of a complex thin filament network and two sets of appendages (Paintrand, M. (1992) J Struct Biol 108: 107-128). The centrosome, a thixotropic blob, is believed to play a key role in the temporal and spatial distribution of the interphasic and mitotic microtubule network and is regarded as a major determinant of the overall organization of the cytoplasm and of the fidelity of cell division (Hsu, L. C. and White, R. L. (1998) Proc Natl Acad Sci USA 27; 95(22): 12983-8). Cytoplasmic organization, cell polarity and the equal partition of chromosomes into daughter cells at the time of cell division, once and only once in each cell cycle, are all ensured through the actions of tightly regulated centrosomal function (Tanaka, T., et al., (1999) Cancer Res 59(9): 2041-4). Centrosome association occurs throughout the mammalian cell cycle, including all stages of mitosis, and determines the number, polarity, and organization of interphase and mitotic microtubules (Tanaka, T., et al., (1999) Cancer Res 59(9): 2041-4; Pihan, G. A., et al., (1998) Cancer Res 58(17): 3974-85). The main function of the centrosome is the nucleation of microtubules and the formation of bipolar spindles (Tanaka, T., et al., (1999) Cancer Res 58(17): 3974-85). Centrosomes and their associated microtubules direct events during mitosis and control the organization of animal cell structures and movement during interphase. The microtubule nucleating ability of centrosomes of tissue sections retain even after several years of storage as frozen tissue blocks (Salisbury, J. L., et al., (1999) J Histochem Cytochem 47(10):1265-74).
Malignant tumors generally display abnormal centrosome profiles, characterized by an increase in size and number of centrosomes, by their irregular distribution, abnormal structure, aberrant protein phosphorylation, and by increased microtubule nucleating capacity in comparison to centrosomes of normal tissues (Lingle, W. L. et al., (1998) Proc Natl Acad Sci USA 95(6): 2950-5; Sato. N., et al., (1999) Clin Cancer Res 5(5):963-70; Pihan, G. A., et al., (1998) Cancer Res 58(17):3974-85; Carroll, P. E., et al., (1999) Oncogene 18(11): 1935-44; Xu, X., et al., (1999) Mol Cell 3(3):389-95; Brinkley, B. R., et al., (1998) Cell Motil Cytoskeleton 41(4):281-8; Doxsey, S. (1998) Nat Genet 20(2):104-6; Kuo, K. K., et al., (2000) Hepatology 31(1):59-64). Among the abnormalities, centrosome hyperamplification is found to be more frequent in a variety of tumor types (Carroll, P. E., et al., (1999) Oncogene 18; 18(11):1935-44; Hinchcliffe, E. H., et al., (1999) Science 283(5403):851-4; Xu, X., et al., (1999) Mol Cell 3(3):389-95; Weber, R. G., et al., (1998) Cytogenet Cell Genet 83:266-269). Although the precise mechanisms by which the centrosomes are (up) regulated during cell cycle are largely unknown, the over-expression of centrosomal kinases or the lack of tumor suppressor genes are observed universally in malignant tumors (Carroll, P. E., et al., (1999) Oncogene 18; 18(11):1935-44; Mussman, J. G., et al., (2000) Oncogene 23; 19(13):1635-46; Zhou, H., et al., (1998) Nat Genet 20(2): 189-93).
Except for the known keyproteins, such as SKP1p, cyclin-dependent kinase 2-cyclin E (Cdk2-E) (Hinchcliffe, E. H., et al., (1999) Science 283(5403): 851-4), kendrin (Flory, M. R., et al., (2000) Proc Natl Acad Sci USA 23; 97(11):5919-23), protein kinase C-theta (Passalacqua, M., et al., (1999) Biochem J 337(Pt 1): 113-8), and EB1 protein, a variety of new cell cycle-regulated kinases or tumor suppressor genes are found to be located in or to be core components of the centrosome. They include Nek2 (Fry, A. M., et al., (1999) J Biol Chem 274(23): 1304-10), protein kinase A type II isozymes (Keryer, G., et al., (1999) Exp Cell Res 249(1):131-146), heat shock Cognate 70 (HSC70) (Bakkenist, C. J., et al., (1999) Cancer Res 59(17):4219-21), PH33 (Nakadai, T., et al., (1999) J Cell Sci 112 (Pt9): 1353-64), AIKs (Kimura, M., et al., (1999) J Biol Chem 274(11)7334-40), human SCF (SKP2) subunit p19(SKP1) (Gstaiger, M., et al., (1999) Exp Cell Res 247(2)554-62), STK15/BTAK (Zhou, H., et al., (1998) Nat Genet 20(2): 189-93), C-Nap1 (Fry, A. M., et al., (1998) J Cell Biol 274(23): 1304-10), Tau-like proteins (Cross, D., et al., (1996) Exp Cell Res 229(2):378-87), cyclin E (Carroll, P. E., et al., (1999; Mussman, J. G., et al., (2000) Oncogene 23; 19(13): 1635-46), retinoblastoma protein pRB and BRCA1 (Hsu, L. C., et al., (1998) Proc Natl Acad Sci USA 95(22):12983-8; Carroll, P. E., et al., (1999) Oncogene 18; 18(11): 1935-44). These proteins are required in the initiation of DNA replication and mitotic progression (Gstaiger, M., et al., (1999) Exp Cell Res 15; 247(2):554-62).
As with most biological processes and particularly with the processes of cell cycle control and signal transduction, the story is more complicated than appears at first sight. It is likely that the proteins or kinases identified to be associated with centrosome dysfunction are only the couple of many in a complex pathway (or parallel pathway) that controls centrosome assembly and function. Support for this idea comes from other new molecules that were reported lately. For example, BTAK/AIK1 (Tanaka, T., et al., (1999) Cancer Res 59(9): 2041-4), AIK3 (Kimura, M., et al., (1999) J Biol Chem 274(11):7334-40), Mdm2 (Carroll, P. E. et al., (1999) Oncogene 18; 18(11): 1935-44) and STK15/BTAK (Zhou, H., et al., (1998) Nat Genet 20: 189-193) are reported to be associated with centrosome dysfunction. The changes such as the loss of p53 tumor suppressor protein and/or the overexpression of these centrosome kinases may cause abnormal centrosome function, abnormal spindle formation, and chromosome segregation (Carroll, P. E. et al., (1999) Oncogene 18; 18(11):1935-44; Zhou, H., et al., (1998) Nat Genet 20:189-193; Tanaka, T., et al., (1999) Cancer Res 59: 2041-44).
It is therefore not difficult to envision how hard could it be to control the aggressive division of cancer cells by inhibiting one or some group of molecules. There are no methods or agents that have been reported to choose the centrosome as a target for this purpose.
Microtubules, a filamentous system, are linear polymers of alpha-and beta (the beta 1, beta2, and beta4 isotypes)-tubulin heterodimers. Except for being a frame of cellular membrane and organelles, microtubules are involved in diverse cellular functions ranging from mitosis to cell motility to signal transduction. Microtubules are the major constituents of mitotic spindles, which are essential for the separation of chromosomes during mitosis (Shan, B., et al., (1999) Proc Natl Acad Sci USA 96(10):5686-5691). They are nucleated by the centrosome through the kinetochores of the centrosome. The spindle is a microtubule-based superstructure that assembles during mitosis to separate replicated DNA. Chromosome attachment to and movement on the spindle is intimately tied to the dynamics of microtubule polymerization and depolymerization. The sister chromatid pairs must maintain a stable attachment to spindle microtubules as the microtubules interconvert between growing and shrinking states. Drugs that are currently used in cancer therapy were designed to perturb microtubule shortening (depolymerization) or lengthening (polymerization) (Compton, D. A., et al., (1999) Science 286:913-914). Unfortunately, a variety of these drugs (such as paclitaxel, docetaxel, etoposide, vincristine, vinblastine, and vinorelbine) are limited by the fact that they all share a common mechanism of action: They bind to tubulin, the molecule of which microtubules are composed, and arrest cells in mitosis by inhibiting spindle assembly (Compton, D. A., et al., (1999) Science 286:313-314). Most recently, some agents (such as monastral) were found, in the in vitro studies, to be able to inhibit mitosis by blocking the function of essential proteins (Mayer, T. U., et al., (1999) Science 286: 971-974). However, proteins involved in the assembly and the maintenance of the mitotic spindle may be tremendous. For example, one class of such proteins is the family of mitotic kinesins, a subset of the kinesin superfamily (Mayer, T. U., et al., (1999) Science 286: 971-974). This superfamily contains over 100 proteins. In addition, as described previously, many key proteins are located in or are the components of centrosome and/or microtubules. Targeting one specific protein out of this tremendous number of proteins is hardly likely to bring a satisfying inhibition of cell mitosis. The consequent problem will inevitably be the development of a variety of resistances as seen with other drugs due to the strong regulating ability of the cancer cells.
Other cytoskeletons such as membrane skeleton, microvilli, cilia, flagella, microfilaments, actin filaments, contractile ring, and intermediate filaments are all important in the organization of the cytoplasm and of the fidelity of cell division.
Except for centrosome and microtubules, other cell organelles or cellular sub-organelles such as mitochondrion, chromosomes, chromatin, nuclei, nuclear matrix, nuclear lamina, core filaments, nuclear envelope (NEs), nuclear pore complexes (NPCs), nuclear membrane, centrioles, pericentriolar materials (PCM), mitotic spindle, spindle pole bodies (SPBs), contractile rings, proteasomes, telomere, plasma membranes, Golgi complexes, Golgi apparatus, endoplasmic reticulum (ER), lysosomes, endosomes, peroxisomes, phagosomes, ribosomes, are all important in maintaining a cell's life. The endoplasmic reticulum, e.g., is the site of synthesis and maturation of proteins.
In the past decades, almost all anti-cancer techniques have focused on the inhibition of the elevated cancer products or the oncogenes that code these products. As the powerful regulating ability and self-defense mechanisms of the aggressively growing cancer cells (Kong, Q. and Lillehei, K. O. (1998) Med Hypotheses 51: 405-409) are greatly ignored, a satisfying outcome following traditional therapies has never been obtained. The commonly recognized reason is likely the development of a variety of resistances prior to recurrence. Thus, there is a need for the development of novel, more effective cancer therapies.